In normal cells, a low mutation rate ensures genetic stability and this depends on effective DNA repair mechanisms for repairing the many accidental changes that occur continually in DNA.
However, during the generation of antibodies, point mutations occur within the V-region coding sequence of the antigen receptor loci and the rate of mutation observed, called somatic hypermutation, is about a million times greater than the spontaneous mutation rate in other genes. The antigen receptor loci are the only loci in human cells that undergo programmed genetic alterations. However, the mechanisms that allow the nucleotide changes to be controlled and targeted to the DNA of a precisely specified part of the genome in this way is not known.
Functional antigen receptors are assembled by RAG-mediated gene rearrangement and the isotype switch from IgM to IgG, IgA and IgE is effected by class switch recombination. Aberrant forms of RAG-mediated gene rearrangement and class switch recombination have been shown to underpin many of the chromosomal translocations associated with lymphoid malignancies. In the case of somatic hypermutation, it was proposed several years ago by Rabbitts et al (1984 Nature 309, 592-597) that the chromosomal translocations which bring the c-myc protooncogene into the vicinity of the IgH locus could make it a substrate for the antibody hypermutation mechanism. Recent evidence using hypermutating cell lines has provided evidence in support of this (Bemark, M. and Neuberger, M. S. 2000 Oncogene 19, 3404-3410). A wider role for aberrant hypermutation came with the finding that several genes apart from the immunoglobulin V genes can (without being translocated into the Ig loci) apparently act as substrates for the antibody hypermutation mechanism in that they exhibit an increased frequency of point mutation in hypermutating B cells. Recent evidence also points to a high frequency of mutations in many B cell tumours and it has been proposed that this is a result of a transient hypermutation phase caused by the antibody hypermutation mechanism. In all these cases, the aberrant mutations are largely at dC/dG residues.
An uncontrolled and enhanced rate of mutation in non-antibody producing cells can also be deleterious. For example, mutations are the hallmark of cancer and the enhanced rate of mutation in cancer cells may explain their capability to continually grow and evade the normal human defences. The “mutator phenotype” hypothesis attributes this phenomenon to an increasing rate of errors in DNA replication as a tumour grows. According to this theory, genes encoding proteins normally interacting with nucleotides such as DNA polymerases and DNA repair enzymes may be faulty in cancer cells and therefore cause subsequent mutations.
In vitro, understanding and harnessing the means for controlling an enhanced rate of mutation can be usefully employed, for example, in generating diversity of gene products such as generating antibody diversity.
Many in vitro approaches to the generation of diversity in gene products rely on the generation of a very large number of mutants which are then selected using powerful selection technologies. For example, phage display technology has been highly successful as providing a vehicle that allows for the selection of a displayed protein (Smith, G. P. 1985 Science, 228, 1315-7; Bass et al. Proteins. 8, 309-314, 1990; McCafferty et al., 1990 Nature, 348, 552-4; for review see Clackson and Wells, 1994 Trends Biotechnol, 12, 173-84). Similarly, specific peptide ligands have been selected for binding to receptors by affinity selection using large libraries of peptides linked to the C terminus of the lac repressor Lacl (Cull et al., 1992 Proc Natl Acad Sci USA, 89, 1865-9). When expressed in E. coli the repressor protein physically links the ligand to the encoding plasmid by binding to a lac operator sequence on the plasmid. Moreover, an entirely in vitro polysome display system has also been reported (Mattheakis et al., 1994 Proc Natl Acad Sci USA, 91, 9022-6) in which nascent peptides are physically attached via the ribosome to the RNA which encodes them.
Artificial selection systems to date rely heavily on initial mutation and selection, similar in concept to the initial phase DNA rearrangement involving the joining of immunoglobulin V, D and J gene segments which occurs in natural antibody production, in that it results in the generation of a “fixed” repertoire of gene product mutants from which gene products having the desired activity may be selected.
Unlike in the natural immune system, however, artificial selection systems are poorly suited to any facile form of “affinity maturation”, or cyclical steps of repertoire generation and development. One of the reasons for this is that it is difficult to generate enough mutations and to target these to regions of the molecule where they are required, so subsequent cycles of mutation and selection do not lead to the isolation of molecules with improved activity with sufficient efficiency.
In vivo, after the primary repertoire of antibody specificities is created by V-D-J rearrangement, and following antigen encounter in mouse and man, the rearranged V genes in those B cells that have been triggered by the antigen are subjected to two further types of genetic modification. Class switch recombination, a region-specific but largely non-homologous recombination process, leads to an isotype change in the constant region of the expressed antibody. Somatic hypermutation introduces multiple single nucleotide substitutions in and around the rearranged V gene segments. This hypermutation generates the secondary repertoire from which good binding specificities can be selected thereby allowing affinity maturation of the humoral immune response. In chicken and rabbits (but not man or mouse) an additional mechanism, gene conversion, is a major contributor to V gene diversification.
Much of what is known about the somatic hypermutation process which occurs during affinity maturation in natural antibody production has been derived from an analysis of the mutations that have occurred during hypermutation in vivo (for reviews see Neuberger and Milstein, 1995 Curr. Opin. Immunol. 7, 248-254; Weill and Reynaud, 1996 Immunol Today 17, 92-97; Parham, 1998 Immunological Reviews, Vol. 162 (Copenhagen, Denmark: Munksgaard)). Most of these mutations are single nucleotide substitutions which are introduced in a stepwise manner. They are scattered over the rearranged V domain, though with characteristic hotspots, and the substitutions exhibit a bias for base transitions. The mutations largely accumulate during B cell expansion in germinal centres (rather than during other stages of B cell differentiation and proliferation) with the rate of incorporation of nucleotide substitutions into the V gene during the hypermutation phase estimated at between 10−4 and 10−3 bp−1 generation−1 (McKean et al., 1984; Berek & Milstein, 1988). However, a greater understanding of the steps involved in these later stages of hypermutation would enable a more diverse range of gene products to be obtained.
All three of the above processes, somatic hypermutation, gene conversion and class-switch recombination, have been shown to depend upon activity of the protein Activation Induced Deaminase (AID) (Muramatsu et al. (1999); Muramatsu M. et al. (2000); Revy, P. et al. (2000); Arakawa, H. et al. (2002); Harris, R. S. et al. (2002); Martin, A. et al. (2002) and Okazaki, I. et al. (2002)) which has been suggested (by virtue of its homology with Apobec-1 (Muramatsu et al. (1999)) to act by RNA editing. However, evidence that the three processes could be initiated by a common type of DNA lesion (Maizels et al. (1995); Weill et al. (1996); Sale et al. (2001); Ehrenstein et al. (1999)) taken with the fact that first phase of hypermutation targets dG/dC (Martin et al. (2002); Rada et al. (1998); Wiesendanger et al. (2000)) has suggested that AID may act directly on dG/dC pairs in the immunoglobulin locus. However, to date, the actual function of AID has not been described.
The AID homologue Apobec-1 has been identified as playing a role in modifying RNA. Apobec-1 is a catalytic component of the apolipoprotein B (apoB) RNA editing complex that performs the deamination of C6666 to U in intestinal apoB RNA thereby generating a premature stop codon. Indeed, the oncogenic activity of Apobec-1, identified by its overexpression in transgenic mice, has previously been attributed to its RNA editing activity acting on inappropriate substrates.
Deamination of cytosine to uracil can occur in vivo at the level of nucleotide and in DNA as well as RNA. In the context of DNA, the low level deamination of cytosine to uracil which takes place spontaneously (and which might be of relatively minor significance when it occurs with free nucleotides or in mRNA) can have major effects, contributing to genome mutation, cancer and evolution (Lindahl, T. (1993) Nature 362, 709-715). However, to date, there is no biochemical evidence that APOBEC family members can trigger such deamination in vitro.